SUMMARY: It is now clear that the ?epitranscriptome,? i.e., the pattern and distribution of regulated nucleotide modifications in mRNA, is dynamic and has functional roles in the brain. We had a founding role in this field by developing the technology for transcriptome-wide mapping of N6-methyladenosine (m6A), which allowed us and others to reveal the transcriptome-wide dynamics of m6A in diverse tissues, signaling and disease contexts. Although m6A is widely studied, it is only one of five abundant methyl modifications that were discovered in mRNA in the 1970's. The other four are part of the ?extended cap structure,? i.e. the cluster of modified nucleotides at the 5' end of mRNA. These are the methyl on the m7G cap, 2'-O-methyl modifications on the ribose of the first and sometimes the second transcribed nucleotides in mRNA, called Cap 1 and Cap 2, respectively. Lastly, if the first transcribed nucleotide of an mRNA is adenosine, it can be methylated one more time after ribose 2'-O-methylation to form dimethyladenosine: N6,2'-O-dimethyladenosine (m6Am). Of these, levels of m6Am and Cap 2 vary between tissues and show evidence for regulation. Nevertheless, little is known about how these dynamic changes in these modifications affects mRNA fates in neurons. In order to uncover their function, we have identified the enzyme that synthesizes m6Am, identified the first m6Am reader and developed a method for mapping Cap 2 throughout the transcriptome. In order to significantly advance our understanding of the dynamics and function of the cap epitranscriptome in neurons, the specific aims of this proposal are: (1) To uncover the mechanism for m6Am dynamics in neural stem cell differentiation. The basis for the dynamic regulation of m6Am is unknown. To understand which mRNAs exhibit dynamic and regulated levels of m6Am, we will use our transcriptome-wide m6Am mapping technique to generate maps of m6Am in different brain regions. We will determine the principles that guide m6Am formation and regulation, and determine if these dynamics are important for neural stem cell differentiation. (2) To determine how m6Am affects the translation and stability of neuronal mRNA. In this aim, we take advantage of our discovery of PCIF1 as the m6Am-forming methyltransferase to uncover the effects of m6Am on translation and mRNA stability. We will also characterize a putative m6Am reader, to identify a mechanism for how m6Am alters neuronal mRNAs. (3) To decipher the dynamics and function of the Cap 2 epitranscriptome. We will obtain the first maps of Cap 2 throughout the brain. Using the Cap 2 maps and depletion of the Cap2- forming methyltransferase, we will determine if Cap 2 is associated with altered mRNA translation, stability, or other aspects of RNA processing. Overall, these studies will allow us to map and determine the role of the ?cap epitranscriptome? in controlling mRNA fate and function in neurons. We expect that this work will stimulate a new area of gene expression regulation research focusing on uncovering how information encoded by methyl modifications in mRNA caps influences mRNA biology.